Use of soluble cytokeratin-1-fragments in diagnostics and therapy

ABSTRACT

Use of novel soluble cytokeratin 1 fragments from body fluids or body tissues as marker peptides for the diagnosis, prognosis and monitoring of the course of inflammations and infections and/or as a target for the therapeutic influencing of the course of inflammations and/or infections.

The present application is a nationalization of PCT Application SerialNo. PCT/EP02/06473, filed Jun. 12, 2002, which claims priority to Germanpriority application Serial No. 101 30 985.6, filed Jun. 27, 2001.

The present invention relates to the use of novel soluble cytokeratin 1fragments in medical diagnosis and therapy. It is based on the detectionfor the first time of the physiological occurrence of certain peptidesin the form of soluble cytokeratin 1 fragments in association with apathological process, in particular in a sepsis or systemic inflammationproduced experimentally in a primate. The term “peptides” is used in thepresent application in the context of a generic term which is intendedto include the condensates of amino acids independently of the length ofthe chain formed, i.e. in particular products which, taking into accounttheir chain length, can be referred to as oligopeptides, polypeptides orproteins.

The present invention has its origin in intensive research work by theApplicant in relation to further improvements of the diagnosis andtherapy of inflammations and infections, in particular of inflammationsof infectious aetiology and sepsis.

Inflammations are defined very generally as certain physiologicalreactions of an organism to different types of external effects, suchas, for example, injuries, burns, allergens, infections bymicroorganisms, such as bacteria and fungi and viruses, to foreigntissues which trigger rejection reactions, or to certain inflammatoryendogenous conditions of the body, for example in autoimmune diseasesand cancer. Inflammations may occur as harmless, localized reactions ofthe body but are also typical features of numerous serious chronic andacute diseases of individual tissues, organs, organ parts and tissueparts.

Local inflammations are generally part of the healthy immune reaction ofthe body to harmful effects and hence part of the life-preservingdefence mechanism of the body. If, however, inflammations are part of amisdirected reaction of the body to certain endogenous processes, suchas, for example, in autoimmune diseases, and/or are of a chronic nature,or if they achieve a systemic extent, as in the case of systemicinflammatory response syndrome (SIRS) or in the case of a severe sepsiscaused by infection, the physiological processes typical of inflammatoryreactions go out of control and become the actual, frequentlylife-threatening pathological process.

It is now known that the origin and the course of inflammatory processesare controlled by a considerable number of substances which arepredominantly of a protein or peptide nature or are accompanied by theoccurrence of certain biomolecules for a more or less limited time. Theendogenous substances involved in inflammatory reactions include inparticular those which may be counted among the cytokines, mediators,vasoactive substances and acute phase protein and/or hormonalregulators. The inflammatory reaction is a complex physiologicalreaction in which both the endogenous substances activating theinflammatory process (e.g. TNF-α) and deactivating substances (e.g.interleukin-10) are involved.

In systemic inflammations, as in the case of a sepsis or of septicshock, the inflammation-specific reaction cascades are spread in anuncontrolled manner over the whole body and become life-threatening inthe context of an excessive immune response. Regarding the currentknowledge about the occurrence and possible role of individual groups ofendogenous inflammation-specific substances, reference is made, forexample, to A. Beishuizen et al., “Endogenous Mediators in Sepsis andSeptic Shock”, Advances in Clinical Chemistry, Vol. 33, 1999, 55–131;and C. Gabay et al., “Acute Phase Proteins and Other Systemic Responsesto Inflammation”, The New England Journal of Medicine, Vol. 340, No. 6,1999, 448–454. Since the understanding of sepsis and related systemicinflammatory diseases, and hence also the recognized definitions, havechanged in recent years, reference is also made to K. Reinhart et al.,“Sepsis und septischer Schock” [Sepsis and septic shock], in:Intensivmedizin, Georg Thieme Verlag, Stuttgart, New York, 2001,756–760, where a modern definition of sepsis is given. In the context ofthe present application, the terms sepsis and inflammatory diseases usedare based on the definitions given in the stated three references.

Whereas at least in Europe the systemic bacterial infection detectableby a positive blood culture long characterized the term sepsis, sepsisis now primarily understood as being systemic inflammation which iscaused by infection but, as a pathological process, has greatsimilarities to systemic inflammations which are triggered by othercauses. Said transformation in the understanding of sepsis has resultedin changes in the diagnostic approaches. Thus, the direct detection ofbacterial pathogens was replaced or supplemented by complex monitoringof physiological parameters and, more recently, in particular by thedetection of certain endogenous substances involved in the sepsisprocess or in the inflammatory process, i.e. specific “biomarkers”.

Of the large number of mediators and acute phase proteins which areknown to be involved in an inflammatory process, the ones which aresuitable for diagnostic purposes are in particular those whoseoccurrence is very specific for inflammatory diseases or certain phasesof inflammatory diseases, whose concentrations change in a dramatic anddiagnostically significant manner and which moreover have thestabilities required for routine determination and reach highconcentration values. For diagnostic purposes, the reliable correlationof pathological process (inflammation, sepsis) with the respectivebiomarker is of primary importance, without there being any need to knowits role in the complex cascade of the endogenous substances involved inthe inflammatory process.

Such an endogenous substance particularly suitable as a sepsis biomarkeris procalcitonin. Procalcitonin is a prohormone whose serumconcentrations reach very high values under the conditions of a systemicinflammation of infectious aetiology (sepsis), whereas it is virtuallyundetectable in healthy persons. High values of procalcitonin are alsoreached in a relatively early stage of a sepsis so that thedetermination of procalcitonin is also suitable for early diagnosis of asepsis and for early distinguishing of a sepsis caused by infection fromsevere inflammations which have other causes. The determination ofprocalcitonin as a sepsis marker is the subject of the publication by M.Assicot et al., “High serum procalcitonin concentrations in patientswith sepsis and infection”, The Lancet, Vol. 341, No. 8844, 1993,515–518; and the patents DE 42 27 454 C2 and EP 0 656 121 B1 and U.S.Pat. No. 5,639,617. Reference is hereby made to said patents and toearly literature references mentioned in said publication forsupplementing the present description. In recent years, the number ofpublications on the subject of procalcitonin has greatly increased.Reference is therefore also made to W. Karzai et al., “Procalcitonin—ANew Indicator of the Systemic Response to Severe Infection”, Infection,Vol. 25, 1997, 329–334; and M. Oczenski et al., “Procalcitonin: a newparameter for diagnosis of bacterial infection in the peri-operativeperiod”, European Journal of Anaesthesiology 1998, 15, 202–209; andfurthermore H. Redl et al., “Procalcitonin release patterns in a baboonmodel of trauma and sepsis: Relationship to cytokines and neopterin”,Crit Care Med 2000, Vol. 28, No. 11, 3659–3663; and H. Redl et al.,“Non-Human Primate Models of Sepsis”, in: Sepsis 1998; 2:243–253; andthe further literature references cited therein, as typical of recentpublished reviews.

The availability of the sepsis marker procalcitonin has givenconsiderable impetus to sepsis research, and intensive efforts are nowbeing made to find further biomarkers which can supplement theprocalcitonin determination and/or are capable of providing additionalinformation for purposes of fine diagnosis or differential diagnosis.The search for potential new sepsis biomarkers is however, complicatedby the fact that frequently very little or nothing is known about theexact function or about the exact reasons for the occurrence of certainendogenous substances which are involved in inflammatory or septicprocesses.

The results of the experimental testing of a fruitful purelyhypothetical approach to the determination of further potential sepsismarkers are to be found in DE 198 47 690 A12 and WO 00/22439. There, itis shown that, in the case of sepsis, not only is the concentration ofthe prohormone procalcitonin increased but also significantly increasedconcentrations can be observed for other substances which may beincluded among the peptide prohormones. While the phenomenon describedis well documented, the causes of the increase in the concentrations ofprohormones in sepsis are still substantially unexplained.

In the present application, results of another, purely experimentalapproach in the search for further inflammation- or sepsis-specificbiomolecules are now reported. These experimental investigations, too,originate in the determination of procalcitonin in relation to systemicinflammatory reactions of infectious aetiology. Thus, it had beenobserved at a very early stage that the procalcitonin is evidently notformed in the same manner in sepsis as when it is a precursor for thehormone calcitonin. Thus, high procalcitonin levels were also observedin patients whose thyroid had been removed. The thyroid therefore cannotbe the organ in which the procalcitonin is formed or secreted duringsepsis. In the publications by H. Redl et al., “Procalcitonin releasepatterns in a baboon model of trauma and sepsis: Relationship tocytokines and neopterin”, Crit Care Med 2000, Vol. 28, No. 11,3659–3663; and H. Redl et al., “Non-Human Primate Models of Sepsis”,Sepsis 1998; 2:243–253, the results of experimental investigations whichare said to be intended for explaining the formation of procalcitonin insepsis are reported. In said work, an artificial sepsis is produced byendotoxin administration to primates (baboons), and the experimentallyproduced states in which the highest procalcitonin concentrations in theblood are reached are determined. A further development of theexperimental animal model described in said work serves, in the contextof the present application, for determining novel endogenoussepsis-specific biomarkers of a peptic or protein nature, the occurrenceof which is characteristic for sepsis or certain forms of sepsis andwhich therefore permit specific diagnosis of sepsis. The primate modelwas chosen because of the very great similarity of the physiology ofprimates and humans and the high cross-reactivity with many therapeuticand diagnostic human reagents.

Since the endogenous substances formed during inflammations are part ofthe complex reaction cascade of the body, not only are such substancesalso of diagnostic interest but attempts are currently also being made,with considerable effort, to intervene therapeutically in theinflammatory process by influencing the formation and/or theconcentration of individual substances of this type, in order to stop asearly as possible the systemic spread of the inflammation, which spreadis observed, for example, during sepsis. In this context, endogenoussubstances which have been shown to be involved in the inflammatoryprocess are also to be regarded as potential therapeutic targets.Attempts based on certain mediators of the inflammatory process andintended to have a positive therapeutic influence on said process aredescribed, for example, in E. A. Panacek, “Anti-TNF strategies”, Journalfür Anästhesie und Intensivbehandlung; No. 2, 2001, 4–5; T. Calandra etal., “Protection from septic shock by neutralization of macrophagemigration inhibitory factor”, Nature Medicine, Vol. 6, No. 2, 2000,164–170; or K. Garber, “Protein C may be sepsis solution”, NatureBiotechnology, Vol. 18, 2000, 917–918. These therapeutic approaches areintended to lower the concentrations of inflammation-promotingsubstances or to inhibit the formation of said substances, and to do soin particular with the use of specific antibodies (against TNF-α or MIF;cf. E. A. Panacek, “Anti-TNF strategies”, Journal für Anästhesie undIntensivbehandlung; No. 2, 2001, 4–5; T. Calandra et al., “Protectionfrom septic shock by neutralization of macrophage migration inhibitoryfactor”, Nature Medicine, Vol. 6, No. 2, 2000, 164–170) or to increasethe concentration of endogenous substances which have an inhibitoryeffect in the inflammation cascade (Protein C; K. Garber, “Protein C maybe sepsis solution”, Nature Biotechnology, Vol. 18, 2000, 917–918). Thelast-mentioned publication gives an overview of such attempts to have atherapeutic influence on the inflammatory process by influencing theselected endogenous target molecules, which attempts have unfortunatelygenerally met with little success to date. In view of the ratherdisappointing therapeutic approaches to date, there is great interest inidentifying further endogenous biomolecules which are as inflammation-or sepsis-specific as possible and which, as therapeutic targets, alsoopen up new prospects for success in fighting inflammation.

The present invention provides novel soluble peptide fragments which areformed in primates and humans during inflammations caused by infectionand are suitable both for inflammation diagnosis and/or sepsis diagnosisand as novel therapeutic targets.

The present invention discloses soluble cytokeratin 1 fragments which,on the basis of their specific occurrence after artificial sepsistriggering by endotoxin administration to primates, have proved to besepsis-specific or inflammation-specific human peptides.

As will be explained in more detail below in the experimental section,the invention is based on the fact that, after experimental triggeringof an artificial sepsis in baboons by endotoxin administration (LPS fromSalmonella Typhimurium) and working up of liver tissue of the treatedanimals by gel electrophoresis, a peptide or protein productidentifiable only in the treated animals was found. This specificproduct was isolated from the electrophoresis gel and investigated bymass spectrometry in a manner known per se.

A first partial sequence of the isolated, trypsin-digested protein spotof 12 amino acids with a mass m/z of 692.39 (SEQ ID NO:1) and a secondpartial sequence of 11 amino acids with a mass m/z of 633.4 (SEQ IDNO:2) could be unambiguously identified, and the sequences could beidentified as fragments of the known but essentially completelyinsoluble cytoskeleton protein cytokeratin 1 (SEQ ID NO:3; cf. L.Johnson et al., Structure of a gene for the human epidermal 67-kDakeratin; Proc. Natl. Acad. Sci. U.S.A.; 82:1896–1900, (1985); databaseNiceProt View of SWISS-PROT: Accession number P04264) by comparison ofthe sequences of these partial peptides with the data of a humandatabase with known protein sequences. The two fragments according toSEQ ID NO:1 and SEQ ID NO:2 correspond to the sequence of the aminoacids 185–196 and 277–287, respectively, of the complete cytokeratin 1.A further fragment of the mass spectrum with a mass m/z (z=1) of 999.49corresponds to a fragment of 9 amino acids. (SEQ ID NO:4; calculatedmass 999.45), which corresponds to the partial sequence of the aminoacids 289–297 of the complete cytokeratin 1 (SEQ ID NO:3).

From these results it can be safely concluded that the peptide isolatedfrom the electrophoresis gel in the form of a cytokeratin 1 fragmentcomprises a sequence of amino acids 185–297 (SEQ ID NO:5) ofcytokeratin 1. However, the fragment corresponding to this sequence (SEQID NO:5) has only a molecular weight of 13615, while the molecularweight determined by gel electrophoresis for the fragment found was15700±500 Dalton. Fragments which are to be regarded as solublecytokeratin 1 fragments according to the invention are therefore inparticular those in which the fragment 185–297 (SEQ ID NO:5) has beenlengthened at one or both of its ends by up to 20 amino acidsaltogether.

The identification of a certain soluble cytokeratin 1 fragment formedduring sepsis in the liver is of considerable scientific, diagnostic andtherapeutic interest.

Cytokeratin 1 is a protein from the group consisting of the structuralproteins (scleroproteins) which are referred to as cytokeratins or“soft” keratins and, as components of the cytoskeleton, form theso-called intermediate filaments (IF). They impart stability of shape tothe cell and are distinguished by high mechanical and chemicalstability. Under customary physiological conditions, they, like allkeratins, are stable to proteases, and there are only a few organisms,such as, for example, the clothes moth and the fungus Tritirachiumalbum, which have enzyme systems which are capable of degrading keratinsand using them as a source of nutrition.

Cytokeratins have a structure comprising an α-helical central section ofabout 310–315 amino acids and end sections adjacent thereto. Thecytokeratins can be assigned to two types on the basis of the amino acidsequences occurring in them and their charge. The cytokeratins 9–20belong to type 1, which includes acidic proteins, while the cytokeratins1–8 are assigned to type II, which includes basic or neutral proteins.Cytokeratins of type I and of type II occur with formation ofheterodimers in certain pairings which are characteristic of certaintypes of epithelial cells and hence also tissue. In this context, forexample, reference may be made to the review article by R. B. Preslandet al., in: Crit Rev Oral Biol Med, 11(4):383–408 (2000) or M. BishrOmary et al., Keratin Modifications and Solubility Properties inEpithelial Cells and in vitro; in: Subcellular Biochemistry, Vol. 31:Intermediate Filaments, N.Y. 1998, pages 105–150.

A determination of IF proteins, which include cytokeratins, has beencarried out to date predominantly in association with tumour diagnosis,since the identification of certain IF proteins in metastases or tissuelesions makes it possible to assign them to a tissue of origin orprimary tumour.

According to EP-A-0 163 304, such a determination is carried out usingsuitable specific antibodies by a histodiagnostic method.

For simplifying the procedure for determinations of said type, EP-B1-0267 355 proposes subjecting the insoluble IF proteins to a treatmentwhich leads to their degradation, and then determining the solublefragments produced artificially in this manner. It is furthermore statedthat cell lesions may also be accompanied by a proteolytic degradationof insoluble structural proteins, so that, under certain conditions,α-helical fragments may also be found in body fluids.

EP-B1-0 267 356 is related but is not concerned with the directdetermination of soluble fragments of IF proteins but of antibodiesformed against such fragments in serum.

EP-B1-0 337 057 further develops the method from EP-B1-0 267 355 as amethod for identifying the origin of a cell sample or tissue sample, acertain standard for use in the determinations being proposed.

The determination of soluble cytokeratin 19 fragments for differentialdiagnosis, prognosis, therapy efficiency monitoring and follow-upobservation in the care of bronchial carcinomas is also described in P.Stieber, CYFRA 21-1 (Cytokeratin 19 fragments) in: L. Thomas, Labor undDiagnose, pages 987–992. The versatility of the cytokeratins, once againin particular the cytokeratins 8, 18 and 19, as tumour markers is alsodiscussed in Torgny Stigbrand, The Versatility of Cytokeratins as TumorMarkers, Tumor Biol 2001, 22:1–3.

All of the above cases are concerned primarily with the determination offragments of the cytokeratins 8, 18 and 19. Among all solublecytokeratin fragments discussed in the literature, there are no solublecytokeratin 1 fragments, and there is also a lack of any informationabout a possible occurrence of such cytokeratin 1 fragments in certainpathological conditions.

It has long been believed that cytokeratin 1—together with cytokeratin10—is found substantially only in a single, immunologically isolatedcell type, namely in suprabasal well differentiated keratinocytes.

An unusual occurrence of cytokeratin 1 and an unexpected involvement ina pathogenic process are described in the papers by Steven D. Lucas etal., Identification of a 35 kD Tumor-Associated Autoantigen in PapillaryThyroid Carcinoma, Anticancer Research 16:2493–2496 (1996), and StevenD. Lucas et al., Aberrantly Expressed Cytokeratin 1, A Tumor-AssociatedAutoantigen in Papillary Thyroid Carcinoma, Int. J. Cancer 73, 171–177(1997) in relation to papillary thyroid carcinoma (PTC). Cytokeratin 1can be detected in the form of a 35 kD fragment in a solubilisate of PTCcells by immunoprecipitation with the aid of patient sera.(Auto)antibodies against cytokeratin 1 are found in the patient sera.There is no mention of soluble cytokeratin 1 fragments from body fluidsand/or body tissues.

The detection, according to the invention, of a soluble cytokeratin 1fragment in the liver of primates in which an artificial sepsis wastriggered by toxin administration, with simultaneous complete absence ofsuch a fragment in otherwise completely identically treated samples ofcontrol animals, is extremely surprising in view of all knowledge todate about the occurrence, the properties and the role of cytokeratin 1.Since the occurrence has been observed only in the treated animals, inparticular a very short time after sepsis triggering by toxinadministration, it is possible to utilize this fact for providing apromising method for diagnosing sepsis, infections and inflammation bydetermination of this fragment formed by the organism.

Since it may be assumed that, due to an increased sepsis- orinflammation-specific protease activity (and/or reduced inhibition ofsuch a protease activity by the protease inhibitors usually involved inthe regulation), the detected fragment is formed by a proteolytic routefrom the complete cytokeratin 1, but not by a modified expression of acorresponding gene, it is to be expected that further solublecytokeratin 1 fragments which correspond to the “remaining” regions ofthe proteolytically degraded complete cytokeratin 1, in particular itsα-helical region, can also be found and, similarly to theabove-mentioned fragments, are suitable for determination. Thedetermination of such further fragments is also to be included expresslyas a variant of the present invention.

Since cytokeratin 1 is found in the respective cells as a rule togetherwith cytokeratin 10, it also appears possible that cytokeratin 10fragments too will be formed in the case of the infection- orinflammation-related increased proteolytic activity of the organism withlesions of cells which contain the two cytokeratins 1 and 10, and, likecytokeratin 1 fragments, can be determined. The cytokeratin 1 fragmentsmay also be present as soluble adducts or aggregates, for example in theform of aggregates with cytokeratin 10 fragments.

It is furthermore within the scope of the present invention to determinecytokeratin 1 fragments which, in the range of the sequences SEQ IDNO:1, SEQ ID NO:2 and SEQ ID NO:5, exhibit certain deviations from thespecific amino acid partial sequences described, in particular whenindividual, patient-specific deviations or polymorphisms can be observedin the range of said sequences. The agreement with said specificsequences is, however, likely to be higher than 75%, and in particularalso higher than 90% or 95%.

The determination can be effected by any desired suitable detectionmethod, but the determination in a body fluid of a patient by animmunodiagnostic method using suitable selective antibodies appears mostadvantageous from practical points of view.

The fact that a certain soluble fragment of the essentially completelyinsoluble cytokeratin 1 was detectable for the first time onexperimentally triggering sepsis thus provides the possibility ofutilizing such cytokeratin 1 fragments for diagnostic and/or therapeuticpurposes. To this end, cytokeratin 1 fragments can, if required, also bespecifically prepared by methods which are now part of the prior art, bysynthesis, genetic engineering or hydrolysis, in particular proteolysis.

Furthermore, cytokeratin 1 fragments or suitable partial sequencesthereof can be used by known processes of the prior art also forproducing specific polyclonal and in particular monoclonal antibodieswhich are suitable as aids for the diagnostic determination ofcytokeratin 1 fragments in body fluids of a patient and/or also aspotential therapeutic agents. The production of suitable monoclonalantibodies against known peptide partial sequences is now part of thegeneral prior art and need not be described in particular. Furthermore,antibody production using techniques of direct genetic immunization witha corresponding DNA should also be mentioned expressly. It is thereforewithin the scope of the present invention to use, for example, a cDNA ofcytokeratin 1 fragments for immunization, since it has been found in thepast that the spectrum of obtainable antibodies can be extended with theuse of such immunization techniques. However, it is also possible to useknown and commercially available antibodies against cytokeratins.

In the immunological determination of soluble cytokeratin 1 fragments,it is possible in principle to proceed as described, for example, forthe selective procalcitonin determination in P. P. Ghillani et al.,“Monoclonal antipeptide antibodies as tools to dissect closely relatedgene products”, the Journal of Immunology, Vol. 141, No. 9, 1988,3156–3163; and P. P. Ghillani et al., “Identification and Measurement ofCalcitonin Precursors in Serum of Patients with Malignant Diseases”,Cancer Research, Vol. 49, No. 23, 1989, 6845–6851; reference being madeexpressly and additionally also to the immunization techniques describedthere, which represent a possibility for obtaining monoclonal antibodiesalso against partial sequences of cytokeratin 1 fragments. A personskilled in the art can consult relevant standard works and publicationsfor variations of the techniques described and/or further immunizationtechniques and can apply them in context.

Cytokeratin 1 fragments having the partial sequences according to SEQ IDNO:1, SEQ ID NO:2, SEQ ID NO:4 and/or SEQ ID NO:5 or partial peptidesthereof, or cytokeratin 1 fragments which are formed in the proteolyticcleavage of the α-helical part of cytokeratin 1 in addition to the abovedetected and characterized fragment, can serve, on the basis of theavailable results, as specific marker peptides (biomarkers) fordiagnosis, for prognosis and for monitoring of the course ofinflammations and infections (in particular of systemic infections ofthe sepsis type). As in the case of the determination of procalcitonin,the determination of at least the specific soluble cytokeratin 1fragment found can be carried out for the differential early diagnosisor for the diagnosis and for the prognosis, for the assessment of theseverity and for the therapy-accompanying assessment of the course ofsepsis and infections, in such a method the content of a certaincytokeratin 1 fragment being determined in a sample of a biologicalfluid or of a tissue of a patient and the presence of an inflammation,of a severe infection or of a sepsis being concluded from theestablished presence and/or amount of the certain peptide and the resultobtained being correlated with the severity of the sepsis, and thepossible treatments and/or the prospects of the treatment beingestimated.

Cytokeratin 1 fragments (or any DNA segments coding for said fragments)can, however, also be used in preventive medicine or therapy.

The fact that it has recently been found that cytokeratin 1 plays animportant role as a type of surface receptor for the so-called highmolecular weight kinin (HK), whose binding is an important step in thetriggering of the bradykinin cascade, plays an important role fortherapeutic realization of the novel discoveries according to theinvention. In this context, reference is made to the results to be foundin the following papers: (a) Zia Shariat-Madar et al.,Kininogen-Cytokeratin 1 Interactions in Endothelial Cell Biology, TCMVol. 9, No. 8, 1999, 238–244; (b) Zia Shariat-Madar et al., MappingBinding Domains of Kininogens on Endothelial Cell Cytokeratin 1; J.Biol. Chem. 273, No. 11, 7137–7145, 1999; (c) K. Joseph et al., FactorXII-dependent Contact Activation on Endothelial Cells and BindingProteins gC1qR and Cytokeratin 1, Thromb. Haemost. 85:119–124, 2001; (d)K. Joseph et al., Activation of the Kinin-Forming Cascade on the Surfaceof Endothelial Cells, Biol. Chem. Vol. 382:71–75 (2001); (e) A. Kaplanet al., Activation of the Plasma Kinin Forming Cascade along CellSurfaces, Int Arch Allergy Immunol 2001; 124:339–342.

In the abovementioned publication (b), a sequence range which is in theimmediate edge region of the experimentally detected cytokeratin 1fragment with sequence SEQ ID NO:5 was determined as the binding sitefor said HK to cytokeratin 1.

The kinins of the so-called bradykinin cascade have, inter alia,vasodilatory, hypotensive and vascular permeability-increasing effects.Since such symptoms are observed in a sepsis and are among thephysiological processes critical for the patient, it cannot be ruled outthat there is a causal relationship between said clinical symptoms and asudden occurrence of soluble cytokeratin 1 fragments in the blood,triggered by the sepsis and associated with a direct effect on thebradykinin cascade. Both an enhancing effect in the sense of anincreased, no longer localized supply of activating HK receptors and aneffect in the sense of a counteraction, for example by binding anddeactivation of kinins secreted in excess are conceivable, and it isalso conceivable that such effects are sequential in aconcentration-dependent manner.

This makes cytokeratin 1 fragments also potential promising therapeutictargets for the therapy of sepsis and similar severe inflammations. Inthis context, depending on which of the abovementioned effects proves tobe the more important, either soluble cytokeratin 1 fragments in theform of drugs can be administered to a patient suffering from sepsis orat risk from sepsis, or the concentration of such fragments can bereduced by administering binding antibodies or by extracorporeal removalof such fragments through a lavage of the blood or plasmapheresis bymeans of affinity absorption. The kinin cascade can be influencedthereby, which can prove to be life-saving.

If soluble cytokeratin 1 fragments as such are used for therapeuticpurposes in drugs, they can also be synthesized by targeted proteolysisusing suitable endoproteases, in particular with the use of cytokeratin1 isolates or concentrates which can be prepared from readily availablematerials utilizing the known solubility properties of the differentcytokeratins in buffers. If endogenous material of a patient (autologousor autogenous material) is used as starting material, optimum toleranceand efficacy are ensured.

Those molecules which contain the chosen cytokeratin 1 fragment inposttranslational modified form, e.g. in glycoslyated or phosphorylatedform, or in a form substituted by pharmaceutical excipients, e.g.polyethylene glycol radicals, should also be regarded as therapeuticallyusable cytokeratin 1 fragments.

The discovery and the identification of a specific cytokeratin 1fragment are described in more detail below, reference being made to theattached sequence listing. The figures show the following:

FIG. 1 shows views of 2D electrophoresis gels which permit a comparisonof the spot patterns of cytoplasmic liver cell protein of a healthybaboon (A) with the liver cell proteins of a baboon 5 h after a sepsisinduced by LPS administration (B). The arrow indicates the position ofthe sepsis-specific product according to the invention (cytokeratin 1fragment) which is distinguished in diagram (B) by a circle;

FIG. 2 shows the mass spectrum of the trypsin-digested isolated productidentified by 2D gel electrophoresis, and

FIG. 3 a shows the results of a tandem electrophoresis of a selectedpeptide fragment of the trypsin digestion with a charge/mass ratio of692.39; and

FIG. 3 b shows the results of a tandem electrophoresis of a furtherselected peptide fragment of the trypsin digestion with a charge/massratio of 633.38;

FIG. 4 shows the results of the determination of the soluble cytokeratin1 fragment in the sera of 20 sepsis patients in comparison with a groupof 16 control persons (blood donors).

1. Infection Simulation by Endotoxin Administration in an Animal Model(Baboons).

On the basis of the experiments carried out with baboons for thestimulation of procalcitonin secretion by endotoxin injections (cf. H.Redl et al., “Procalcitonin release patterns in a baboon model of traumaand sepsis: Relationship to cytokines and neopterin”, Crit Care Med2000, Vol. 28, No. 11, 3659–3663; H. Redl et al., “Non-Human PrimateModels of Sepsis”, in: Sepsis 1998; 2:243–253), baboons (male, about 2years old, weighing from 27 to 29 kg) were each intravenouslyadministered 100 μg of LPS (lipopolysaccharide from SalmonellaTyphimurium, source: Sigma) per kg body weight. From 5 to 5.5 h afterthe injection, the animals were sacrificed by intravenous administrationof 10 ml of doletal. Within 60 min of their death, all organs/tissueswere dissected and were stabilized by freezing in liquid nitrogen.

During the further processing, 1.5 ml of buffer A (50 mM Tris/HCl, pH7.1, 100 mM KCl, 20% of glycerol) were added to samples of theindividual frozen tissues (1 g) while cooling with nitrogen, and thesamples were pulverized in a porcelain mortar to give a powder (cf. J.Klose, “Fractionated Extraction of Total Tissue Proteins from Mouse andHuman for 2-D Electrophoresis”, in: Methods in Molecular Biology, Vol.112: 2-D Proteome Analysis Protocols, Humana Press Inc., Totowa, N.J.).After subsequent centrifuging for 1 hour at 100,000 g and +4° C., thesupernatant obtained was recovered and was stored at −80° C. untilrequired for further processing.

Using the tissue extracts obtained in this manner, an investigation wasfirst carried out to determine in which of the tissues investigated thelargest amounts of the known sepsis biomarker procalcitonin can beproduced by endotoxin administration. In the determined tissue havingthe highest level of procalcitonin formation, further previouslyunidentified protein products which occurred only after endotoxinadministration were then sought by means of differential proteomeanalysis. For this purpose, tissue samples of untreated baboons wereused as control tissue samples, the sacrificing and obtaining of sampleshaving been effected under conditions identical to those in the case ofthe treated animals.

2. Determination of Baboon Tissues Having the Highest Level ofProcalcitonin Formation After Endotoxin Injection

Samples of the individual tissues were investigated with the aid of animmunoluminometric test which operates (similarly to the LU-MItest® PCTof the Applicant, developed for the determination of humanprocalcitonin) with, on the one hand, an antibody against babooncalcitonin, immobilized on polystyrene tubes, and a monoclonal antibodymarked with an acridinium ester and directed against the N-terminus ofbaboon procalcitonin. With the aid of this test, the contents of baboonprocalcitonin in the individual samples were determined aftercalibration of the test using recombinant human procalcitonin.

The experiments showed that liver tissue gives the largest amount ofprocalcitonin. The protein extracts from baboon liver which wereobtained in the manner described at the outset were therefore used forsearching for novel sepsis-specific biomarkers.

3. Proteome Analysis Using Cytoplasmic Liver Cell Proteins of Baboons.

Cytoplasmic liver cell protein extracts of, on the one hand, healthybaboons (control) and, on the other hand, baboons which had beeninjected with LPS were used in a proteome analysis. In the initialanalytical 2D gel electrophoresis, liver extract containing 100 μg ofprotein was stabilized to 9M urea, 70 mM DTT, 2% ampholyte pH 2–4 andthen separated by means of analytical 2D gel electrophoresis, asdescribed in J. Klose et al., “Two-dimensional electrophoresis ofproteins: An updated protocol and implications for a functional analysisof the genome”, Electrophoresis 1995, 16, 1034–1059. The visualizationof the proteins in the 2D electrophoresis gel was effected by means ofsilver staining (cf. J. Heukeshoven et al., “Improved silver stainingprocedure for fast staining in Phast-System Development Unit. I.Staining of sodium dodecyl gels”, Electrophoresis 1988, 9, 28–32).

For evaluation, the protein spot patterns of the samples of untreatedanimals were compared with the protein spot patterns which resulted fromliver tissue samples of treated animals. Substances which occurred in nocontrol sample but additionally in all treated animals were selected forfurther analytical investigations. FIG. 1 shows a comparison of the 2Delectrophoresis gels for a control sample (A) and a sample of a treatedanimal (B), the additional protein spot in (B) corresponding to thenovel soluble cytokeratin 1 fragment, the position of which is singledout by an arrow and a circle.

The novel specific proteins identified in the protein spot pattern ofthe analytical 2D gel electrophoresis were then prepared by means ofpreparative 2D gel electrophoresis using 350 μg of protein (once againcf. (10)). In the preparative 2D gel electrophoresis, the staining waseffected by means of Coomassie Brilliant Blue G250 (cf. V. Neuhoff etal., “Improved staining of proteins in polyacrylamide gels includingisoelectric focusing gels with clear background at nanogram sensitivityusing Coomassie Brilliant Blue G-250 and R-250”, Electrophoresis 1988,9, 255–262).

The protein spots preselected for the further analysis were cut out ofthe gel, using the method which is described in A. Otto et al.,“Identification of human myocardial proteins separated bytwo-dimensional electrophoresis using an effective sample preparationfor mass spectrometry”, Electrophoresis 1996, 17, 1643–1650,trypsin-digested and then analyzed by mass spectroscopy, in particularwith the use of mass spectrometric investigations as described anddiscussed, for example, in G. Neubauer et al., “Mass spectrometry andEST-database searching allows characterization of the multi-proteinspliceosome complex”, in: nature genetics vol. 20, 1998, 46–50; J.Lingner et al., “Reverse Transcriptase Motifs in the Catalytic Subunitof Telomerase”, in: Science, Vol. 276, 1997, 561–567; M. Mann et al.,“Use of mass spectrometry-derived data to annotate nucleotide andprotein sequence databases”, in: TRENDS in Biochemical Sciences, Vol.26, 1, 2001, 54–61. After an ESI (ElectroSprayIonization), thetrypsin-digested samples were subjected to tandem mass spectrometry. AQ-TOF mass spectrometer having a so-called nanoflow-Z-Spray ion sourcefrom Micromass, UK, was used. The procedure corresponded to the workinginstructions of the equipment manufacturer.

4. Identification of a Soluble Cytokeratin 1 Fragment

As shown in FIGS. 1(A) and 1(B), liver cell extracts of baboons to whichan LPS injection had been administered contained, inter alia, a novelprotein for which a molecular weight of about 15,700±500 Dalton wasestimated on the basis of the gel electrophoresis data in comparisonwith marker substances having a known molecular weight, while anisoelectric point of from about 5.5 to 6.5 was estimated from therelative position of the protein from the first dimension.

This protein was analyzed as above by mass spectrometry, and it waspossible to assign to the two trypsin fragments according to FIGS. 3 aand 3 b the amino acid sequences SEQ ID NO:1 and SEQ ID NO:2, whichproved to be partial sequences of the known sequence of cytokeratin 1(SEQ ID NO:3; cf. L. Johnson et al., Structure of a gene for the humanepidermal 67-kDa keratin; Proc. Natl. Acad. Sci. U.S.A.; 82:1896–1900,(1985); database NiceProt View of SWISS-PROT: Accession number P04264).The two fragments according to SEQ ID NO:1 and SEQ ID NO:2 correspond tothe sequence of the amino acids 185–196 and 277–287, respectively, ofthe complete cytokeratin 1. A further fragment of the mass spectrumhaving a mass m/z (z=1) of 999.49 corresponds to a fragment of 9 aminoacids (SEQ ID NO:4; calculated mass 999.45) which corresponds to thepartial sequence of the amino acids 289–297 of the complete cytokeratin1 (SEQ ID NO:3).

These results permit the safe conclusion that the peptide in the form ofa cytokeratin 1 fragment, isolated from the electrophoresis gel,comprises the sequence of the amino acids 185–297 (SEQ ID NO:5) ofcytokeratin 1. However, the fragment corresponding to this sequence (SEQID NO:5) has only a molecular weight of 13,615, while the molecularweight of the fragment found, determined by gel electrophoresis, was15,700±500 Dalton. Consequently, in particular those fragments in whichthe fragment 185–297 (SEQ ID NO:5) has been lengthened at one or both ofits ends by up to 20 amino acids altogether are to be regarded assoluble cytokeratin 1 fragments according to the invention.

5. Determination of the Soluble Cytokeratin 1 Fragment in Sera

The serum concentrations of the above-mentioned soluble cytokeratin 1fragments were determined in 20 sera of sepsis patients in whom highvalues for the sepsis marker procalcitonin (PCT) had been found. In 95%of the sepsis sera, greatly increased concentrations (more than 3 ng/ml)were found.

For the exploratory determinations in sepsis sera, a competitiveluminescence immunoassay specially developed for this purpose was used,in which immunoassay sheep antibodies against a peptide which compriseda partial sequence of the cytokeratin 1 fragment, which included theamino acids 214 to 229 of SEQ ID NO:3, were used. The synthetic peptideused for obtaining antibodies and as competitor is commerciallyavailable under the name peptide PLY17 (Jerini BioTools GmbH).

The following procedure was used for carrying out the determination:

Polystyrene tubes (from Greiner) were coated with 100 ng of peptide(PLY17; SEQ ID NO:1) in 300 μl of PBS. After incubation for 20 hours atroom temperature, washing was effected with 2×4 ml of PBS, containing 1%of BSA. The peptide-coated tubes were then used as a solid phase forcarrying out the subsequent measurements in which the immobilizedpeptides and the cytokeratin 1 fragments from the sample competed for asheep antibody against the above-mentioned partial peptide sequence,which antibody had been added in the form of an antiserum.

The following procedure was used for the measurement:

-   1. pipette 100 μg of the sample (sepsis serum or control serum or    calibrator solution) into the above-mentioned tubes;-   2. pipette 200 μl of antiserum (diluted 1:5000 with PBS);-   3. incubate for 3 h at room temperature with shaking;-   4. wash the unbound antibody out of the tubes (filled 4× with 1 ml    of PBS and decanted);-   5. add an acridinium ester-marked donkey anti-sheep antibody    (B.R.A.H.M.S Diagnostica) in 300 ml of PBS, 1% of BSA for marking    the solid phase-bound antibodies;-   6. after incubation for 2 h at room temperature, remove the unbound    marking antibody and wash as under 4;-   7. measure the acridinium ester bound to the solid phase in a known    manner by means of a luminometer (from Berthold).

For the preparation of a calibration curve, solutions containing knownamounts of the above-mentioned synthetic peptide were used, and theconcentrations of the soluble cytokeratin 1 fragment were determined bycomparison of the measured values for the sepsis sera with thecalibration curve.

A graph of the measured results is shown in FIG. 4. Even with theprovisional, very simple and insensitive competitive measurementprocedure described, very good sensitivity of the determination of thecytokeratin 1 fragments in the case of sepsis is evident.

The cytokeratin 1 fragment found is to be designated as a novel protein(or peptide) whose presumably proteolytic formation was observed for thefirst time after contact of the primate organism with the endotoxinsadministered. It has to date only been possible to speculate about apossible natural role of the fragment. However, its identification forthe first time and the documented high specificity make it a promisingdiagnostic target and a novel interesting target for therapeuticintervention.

It is furthermore within the scope of the present invention to use thecytokeratin 1 fragment identified or a related fragment, optionally alsoa part-fragment, as pharmaceutical active substances. The inventionaccordingly also relates to pharmaceutical compositions which contain,as the actual active substance, one of the peptides according to theinvention or antibodies produced against these peptides and prepared foradministration to patients together with a suitable pharmaceuticalcarrier.

1. A method for diagnosis of systemic inflammation and/or systemicinfection in an animal or patient, comprising testing a biologicalsample from said animal or patient for the presence of a solublecytokeratin 1 fragment or soluble cytokeratin 1 fragments, wherein thesoluble cytokeratin 1 fragment consists of the amino acid sequence ofamino acids 151–510 of the complete amino acid sequence of cytokeratin 1(SEQ ID NO: 3) or the soluble cytokeratin 1 fragment has a molecularweight, as determined by gel electrophoresis of, 15,700±500 Dalton, hasan amino acid sequence comprising amino acids 185–297 (SEQ ID NO: 5) ofthe complete amino acid sequence of cytokeratin 1 (SEQ ID NO: 3) and oneor more additional amino acids at one or both ends of SEQ ID NO: 5 for atotal of up to 20 adjacent amino acids of the complete amino acidsequence of cytokeratin 1 and wherein the presence of said fragment orfragments is indicative of the presence of sepsis, sepsis-like systemicinflammation and/or infection.
 2. The method according to claim 1,wherein said biological sample is a biological fluid or a tissue sample.3. The method according to claim 1, wherein the soluble cytokeratin 1fragment has a molecular weight, as determined by gel electrophoresis,of 15,700±500 Dalton, has an amino acid sequence of amino acids 185–297(SEQ ID NO: 5) and one or more additional amino acids at one or bothends of SEQ ID NO: 5 for a total of up to 20 adjacent amino acidswherein said amino acid sequence corresponds to the complete amino acidsequence of cytokeratin 1 or a sequence of at least 90% identity withthe complete amino acid sequence of cytokeratin 1 and wherein saidfragment is detectable by an antibody specific for an epitope of apeptide consisting of amino acids 214 to 229 of SEQ ID NO:
 3. 4. Themethod according to claim 1, wherein the presence of a solublecytokeratin fragment or soluble cytokeratin fragments is determined byan immunodiagnostic method of determination.
 5. The method according toclaim 3, wherein the soluble cytokeratin 1 fragment determined has theamino acid sequence of amino acids 185–297 (SEQ ID NO: 5) of thecomplete amino acid sequence of cytokeratin 1 (SEQ ID NO: 3) and up to20 adjacent amino acids of the complete amino acid sequence ofcytokeratin
 1. 6. The method according to claim 5, wherein the solublecytokeratin 1 fragment determined has the amino acid sequence of aminoacids 185–297 (SEQ ID NO: 5) of the complete amino acid sequence ofcytokeratin 1 (SEQ ID NO: 3).
 7. The method according to claim 1,wherein said soluble cytokeratin 1 fragment or fragments are present ata concentration of at least 3 ng/ml.